Fig. 4
The PPQY motif in NF1 interacted directly with the WW domains of YAP1. A Alternatively two NF1 spliced transcript variants encoding different isoforms have been presented in this diagram, and the sites of qRT-PCR primers were marked in different colors. B NF1-2 was the main isoform in NOZ and EH-GB1, according to the detection of different isoforms of NF1 by qRT-PCR. C Domain architectures of YAP1 and the amino-acid residue numbers were indicated. The domain names were abbreviated within the respective colored regions: CC (Coiled coil, sky blue), WW1 (Green yellow), WW2 (Medium purple), and TAD (Transactivation domain, light brown). Sequence alignment of WW domains in YAP1 was presented by Clustal W. D Dissociation constant between PPQY and WW domains in YAP1 as analyzed by ITC assay. Quantification of the dissociation constant (KD) of the interaction between NF1PPQY and YAP1WW, as measured by an isothermal titration calorimetry assay. The KD value was indicated. E NF1PPQY directly interacted with WW1 and WW2, as shown by GST Pull-down assay. SDS–PAGE showed pull-down results of the NF1 and YAP1 by either GST-tagged NF1PPQY or NF1AAAA, respectively, compared to a GST control. 1 μg of each input protein was loaded on the left. Positions of molecular weight markers are indicated in kDa. F Left: Cartoon representation of NF1PPQY (blue) in complex with YAP1WW1 (green) or YAP1WW2 (purple). Right: Electrostatic potential surface analysis showed the hydrophobic interactions between YAP1 WW1 and the PPQY motif